White Spot Syndrome Virus (WSSV) is a major viral disease in shrimp in large areas of Southeast Asia. The virus has a wide host range among crustaceans (Flegel, 1997) and there is little genetic variation among isolates (Lo et al, 1999). Electron microscopy (EM) studies showed that the virions are enveloped and have a rod to bullet shaped appearance of about 275 nm in length and 120 nm wide with a tail-like appendage at one end. Nucleocapsids, which have lost their envelope, have a crosshatched appearance and a size of about 300 nm×70 nm (Wongteerasupaya et al., 1995). This virion morphology, its nuclear localization and its morphogenesis are reminiscent of baculoviruses in insects (Durand et al., 1997). Originally, WSSV has been classified as an unassigned member of the Baculoviridae family (Francki et al., 1991) hence the virus has been referred to as Systemic Ectodermal Mesodermal Baculo virus (SEMBV) or White Spot Baculo virus (WSBV). At present WSSV is no longer accepted into this family (Murphy et al., 1995) due to lack of molecular information. The double stranded viral DNA has a size of well over 200 kb as derived from restriction endonuclease analysis (Yang et al., 1997).
An outbreak of WSSV in cultured shrimp in Southeast Asia causes mass mortality among the shrimp. The disease is characterized by white spots on the carapace, appendages and cutucie and reddish coloration of the hepatopancreas of the shrimp. The infected shrimps show signs of lethargy and a rapid reduction in food consumption and within 3 to 5 days these shrimp normally die. An outbreak of WSSV leads to heavy losses in the industry of cultured shrimp and as a consequence there is a strong need for vaccines that can protect against WSSV infections. The identification and characterization of major structural WSSV proteins that can be used in such a vaccine would provide the means to develop such vaccines.
Four major proteins of WSSV have been identified which have been designated VP28 (28 kDa), VP26 (26 kDa), VP24 (24 kDa) and VP19 (19 kDa due to their molecular weight estimated from their mobility in Coomassie Brilliant Blue-stained SDS-PAGE gels. VP26 and VP24 are nucleocapsid proteins, whereas VP28 and Vp19 are envelope proteins. The N-terminal amino acid residues of the WSSV proteins were obtained by protein sequencing, and were used to identify their genes (vp28, vp26, vp24, vp19, respectively) on the WSSV genome. The open reading frame (ORF) of vp26 comprises 555 nucleotides and is depicted in FIG. 2b (SEQ ID NO.1) together with the deduced amino acid sequence of VP26, which is depicted as an 184 amino acid residues sequence (SEQ ID NO. 3) in FIG. 2b. A second open reading frame of vp26 comprises 612 nucleotides and is depicted in SEQ ID NO. 9 together with the deduced amino acid sequence consisting of 204 residues, which is separately depicted as SEQ ID NO. 10. The open reading frame of vp28 comprises 615 nucleotides (SEQ ID NO. 2) and is depicted in FIG. 2c together with the deduced amino acid sequence (SEQ ID NO.4). The deduced amino acid sequence of VP28 is 204 amino acids. Both VP26 and VP28 contain a putative transmembrane domain at the N-terminus and many putative N- and O-glycosylation sites. The ORF of the genes vp26 and vp28 coded for proteins with a theoretical size of 20 kDa and 22 kDa respectively. The theoretical amino acid sequence of VP26 and VP28 was confirmed by direct protein sequencing. The theoretical sizes of VP26 and VP28 differ 6 kDa from the size estimated by their mobility in Coomassie Brilliant Blue-stained SDS-PAGE gels. This size difference could be explained by posttranslational modifications such as glycosylation, phosphorylation, etc. The N-terminal amino acid sequence of VP24 and the partial amino acid sequence of VP19 are depicted in SEQ ID NOS. 5 and 6 respectively. The complete open reading frame of VP24 comprises 627 nucleotides and is depicted in SEQ ID NO. 11 together with the deduced amino acid sequence of VP19. The deduced amino acid sequence of VP24 has 208 residues and is separately depicted in SEQ ID NO. 12. The four proteins and their respective nucleotide sequences are specific for WSSV.